Dissecting specific Wnt components governing osteogenic differentiation potential by human periodontal ligament stem cells through interleukin-6

Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and β-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues.


Results
Isolated hPDLSCs exhibit MSC-like properties. In two-dimensional (2D) culture, hPDLSCs exhibited a fibroblast-like morphology and tended to adhere to the culture dish (Fig. 1A). Flow cytometry confirmed expression of the MSC-related surface markers CD44, CD73, CD90, and CD105, but not the hematopoietic cell surface marker CD45 (Fig. 1B), in addition to the stemness-related markers REX1, NANOG, and OCT4 as well as the proliferative marker KI67 (Fig. 1C). The colony-forming capability of hPDLSCs was also observed (Fig. 1D,E). Staining and mRNA marker expression demonstrated the multilineage differentiation potential of hPDLSCs toward osteogenic, adipogenic, and chondrogenic lineages ( Fig. 1F-K) (n = 4). Collectively, these results highlight the characteristics shared by MSCs and hPDLSCs.
Physiological and inflammatory levels of IL-6 promote osteogenic differentiation potential of hPDLSCs in vitro. The effects of IL-6 at 10 and 20 ng/mL on the osteogenic differentiation potential of hPDLSCs in vitro were assessed. The results of the cell proliferation and viability assays showed that IL-6 was not cytotoxic ( Fig. 2A). hPDLSCs were maintained in culture medium pre-treated with and without IL-6 at 10 and 20 ng/mL. Proliferation and viability were determined with the alamarBlue assay and staining of viable and dead cells on days 1, 5, and 7. The results of the alamarBlue assay showed no significant difference in cell proliferation on days 1, 5, and 7 between the control group and the groups treated with IL-6 at 10 and 20 ng/mL (Fig. 2B), while the cell viability assay reveled no difference in the populations of viable and dead cells at every time point (Fig. 2C) (n = 4). Since IL-6 at 10 and 20 ng/mL was not cytotoxic, these dosages were used to assess the effects of IL-6 on the osteogenic differentiation potential of hPDLSCs in vitro.
To assess the effect of IL-6 on osteogenic differentiation potential in vitro, hPDLSCs were pre-treated with and without IL-6 at 10 and 20 ng/mL for 24 h and then cultured in osteogenic induction medium (OM) for 21 days (Fig. 3A). The results showed that IL-6 promoted the osteogenic differentiation potential of hPDLSCs in vitro by enhancing alkaline phosphatase (ALP) activity, osteogenic mRNA marker expression, and matrix mineralization. At days 14 and 21, IL-6 at 20 ng/mL significantly enhanced the ALP activity of hPDLSCs during osteogenic induction (Fig. 3B). Additionally, IL-6 treatment significantly upregulated expression of pivotal osteogenic mRNA markers (RUNX2, OSX, COL1, ALP, OCN, and OPN) during osteogenic induction, especially at 20 ng/mL (Fig. 3C). IL-6 at 20 ng/mL also enhanced matrix mineralization of hPDLSCs as compared to the osteogenic control group, which confirmed the results of Von Kossa and Alizarin Red S staining (Fig. 3D) (n = 4). Since, IL-6 at 20 ng/mL significantly enhanced the osteogenic differentiation potential of hPDLSCs in vitro, this dosage was applied in the following experiments.
Wnt, TGF-β1, and Notch signaling may be involved in IL-6-mediated osteogenic differentiation of hPDLSCs in vitro. To further identify potential signaling pathways involved in the regulation of osteogenic differentiation, hPDLSCs pre-treated with IL-6 at 20 ng/mL for 24 h were cultured in OM for 21  www.nature.com/scientificreports/ and the expression levels of specific target genes were measured on days 1, 3, 7, 14, and 21 (Fig. 4A). The results showed that the expression levels of components of the Wnt/β-catenin signaling pathway (LEF1, TCF7, and β-catenin) were significantly upregulated in response to IL-6 at 20 ng/mL as compared to the undifferentiated and osteogenic control groups at almost every time point (Fig. 4B), while expression of the TGF-β1 target genes (BMP-2, TMEFF1, and CXXC5) and Notch target genes (HES1, HEY1, and LFNG) was significantly increased at some, but not all, time points (Fig. 4C,D) (n = 4). The results implicated these signaling pathways in IL-6-mediated osteogenic differentiation of hPDLSCs in vitro.
Canonical and non-canonical Wnt pathways potentially govern IL-6-mediated osteogenic differentiation of hPDLSCs in vitro. Next, hPDLSCs pre-treated with IL-6 at 20 ng/mL were treated for 24 h with the inhibitors Dickkopf-1 (DKK1), SP600125, SB431542, and DAPT (N-[N-(3,5-difluorophenacetyl)l-alanyl]-s-phenylglycinet-butyl ester) specific to the canonical Wnt, non-canonical Wnt, TGF-β1, and Notch pathways, respectively, and then cultured in OM for 21 days (Fig. 5A). The results showed that Dkk-1 and SP600125 significantly suppressed ALP activity at days 14 and 21, while SB431542 and DAPT suppressed ALP activity only at day 21 (Fig. 5B). Interestingly, both Dkk-1 and SP600125 dramatically downregulated expres-  WNT2B, WNT10B, and WNT5A are related to IL-6-mediated osteogenic differentiation of hPDLSCs in vitro. To further determine the relevance of potential signaling pathways on IL-6-mediated osteogenic differentiation, representative components of each pathway were analyzed. Thus, hPDLSCs pretreated with IL-6 at 20 ng/mL were treated with Dkk-1, SP600125, SB431542, or DAPT for 24 h and then cultured in OM for 21 days. Afterward, the mRNA expression levels of specific ligands and transcription factors of the canonical Wnt, non-canonical Wnt, TGF-β1, and Notch pathways were measured at days 1, 3, 7, 14, and 21 (Fig. 6A). The results showed that IL-6 treatment significantly upregulated mRNA expression of some components of the canonical Wnt (WNT2B, WNT10B, LEF1, and β-catenin) and non-canonical Wnt (WNT5A) pathways, while the specific inhibitors Dkk-1 and SP600125 significantly suppressed expression of these markers (Fig. 6B,C). However, this trend was not observed for components of the TGF-β1 and Notch signaling pathways (Fig. 6D,E) (n = 4). These findings suggest that the canonical and non-canonical Wnt pathways participate in IL-6-mediated osteogenic differentiation of hPDLSCs in vitro via the Wnt pathway-specific ligands WNT2B, WNT10B, and WNT5A.
WNT2B, WNT10B, and WNT5A are different key regulators on IL-6-mediated osteogenic differentiation of hPDLSCs in vitro. Gene silencing study. To determine the relevance of WNT2B, WNT10B, or WNT5A in IL-6-mediated osteogenic differentiation of hPDLSCs, each Wnt ligand was transiently knocked down by small-interfering RNA (siRNA) upon IL-6 treatment during osteogenic induction. Then, analyses of osteogenic mRNA marker expression, ALP activity, and matrix mineralization were performed, as illustrated in Fig. 7A. Interestingly, the results showed that silencing of either WNT2B or WNT10B significantly diminished IL-6-induced ALP activity at days 14 and 21, while silencing of WNT5A only suppressed ALP activity (Fig. 7B). Further analyses showed that silencing of only WNT2B, WNT10B, or WNT5A mostly diminished

Recombinant WNT study.
To further confirm the relevance of WNT2B, WNT10B, or WNT5A on IL-6-mediated osteogenic differentiation of hPDLSCs, each recombinant human WNT (rhWNT) was applied upon IL-6 treatment during osteogenic induction. Then, analyses of osteogenic mRNA marker expression, ALP activity, and matrix mineralization were performed as illustrated in Fig. 8A. The results showed that either rhWNT2B or rhWNT10B significantly enhanced IL-6-mediated ALP activity at days 14 and 21 (Fig. 8B). Further analyses The results of the rhWNT study suggest that either WNT2B or WNT10B plays a potential role in IL-6-mediated osteogenic differentiation of hPDLSCs in vitro, while WNT5A may have a comparatively lesser impact.

Stabilization and translocation of β-catenin study.
To explore the roles of WNT2B, WNT10B, and WNT5A along with the relevance of the Wnt/β-catenin-dependent pathway in IL-6-mediated osteogenic differentiation of hPDLSCs, either siRNA or rhWNT of each Wnt ligand was applied upon IL-6 treatment during osteogenic induction. Then, mRNA expression of each Wnt ligand and β-catenin stabilization and translocation were analyzed ( Fig. 9A,B). The results showed that siRNA significantly downregulated expression of WNT2B, WNT10B, and WNT5A at all time points, as compared to the IL-6-treated group, while rhWNT significant upregulated WNT2B and WNT10B at almost all time points. However, as compared to the IL-6 treated group, WNT5A was significantly upregulation only at day 1 ( Fig. 9C-E). These results illustrate the dynamic expression of WNT2B, WNT10B, and WNT5A upon IL-6 treatment. Further analyses of subcellular β-catenin stabilization and translocation showed that, upon stimulation by rhWNT, both rhWNT2B and rhWNT10B, but not rhWNT5A, significantly enhanced cellular stabilization and nuclear translocation of β-catenin at all time points (6, 24, and 48 h) ( Fig. 9F-N, Supplementary Figs. 1A-3B) (n = 4), suggesting that IL-6 employed the canonical Wnt/βcatenin-dependent pathway via either WNT2B or WNT10B to promote the osteogenic differentiation potential of hPDLSCs in vitro. Besides, IL-6 may also employ the non-canonical Wnt pathway via WNT5A. A summary of the Wnt-related signaling pathways governing osteogenic differentiation of hPDLSCs treated with IL-6 is presented in Fig. 10.

Discussion
Homeostasis of alveolar bone and periodontal tissue plays an important role in the progression of oral and periodontal diseases 12,48 . The most important factor influencing homeostasis of these tissues is the state of  www.nature.com/scientificreports/ inflammation 17 . Chronic inflammation that accompanies severe periodontitis can lead to destruction of periodontal tissue and alveolar bone 3 . The inflammatory response within the tissue is dependent on the balance of pro-and anti-inflammatory cytokines 21,23 . In the early stage of inflammation, IL-6 release is promoted by various pro-inflammatory cytokines, including IL-1, IL-3, tumor necrosis factor alpha (TNF-α), interferon gamma, and platelet-derived growth factor 17,18 . Previous studies have reported that IL-1 and TNF-α are associated with bone loss in oral diseases 19,49 . IL-1 stimulates T and B cells to amplify inflammatory responses and promotes macrophage production of receptor activator nuclear factor-kappa B (RANK) and the corresponding ligand RANKL. Binding of RANKL to RANK promotes monocyte/macrophage differentiation toward preosteoclasts, which leads to osteoclastogenesis and the process of bone resorption 17,18 . In addition, TNF-α increases osteoclastic bone resorption and inhibits osteoblast differentiation 19 . To explore the relevance of potential regulating pathways, the effects of IL-6 (20 ng/mL) on specific pathway target genes expressed by hPDLSCs upon inhibition of crucial osteogenic-regulating pathways (canonical Wnt, non-canonical Wnt, TGF-β1, and Notch pathways) were analyzed. Dkk-1, SP600125, SB431542, and DAPT were used to block the canonical Wnt, non-canonical Wnt, TGF-β1, and Notch signaling pathways, respectively. Effects of IL-6 (20 ng/ mL) on specific pathway target genes expressed by hPDLSCs upon treatment with each specific inhibitor of the (B) canonical Wnt, (C) non-canonical Wnt, (D) TGF-β1, and (E) Notch signaling pathways. Gene expression was quantified by RT-qPCR (n = 4). Superscript letters indicate significant differences vs. the undifferentiated control ( a ), osteogenic control ( b ), and osteogenic induction (IL-6 at 20 ng/mL) upon specific inhibitor treatment ( c ) (p < 0.05). www.nature.com/scientificreports/ On the other hand, IL-6 plays a biphasic role in bone tissue homeostasis 50 : inhibition of osteoclastogenesis 51,52 and initiation of osteolysis and osteoporosis 53,54 . Expression of IL-6 is upregulated in inflamed periodontal tissues 55,56 . Interestingly, expression of IL-6 has also been detected in other dental tissues and cells 57,58 , suggesting that IL-6 is closely related to the homeostatic capacity of oral and periodontal tissues. Therefore, comprehensive understanding of the role of IL-6 is key to address the destruction of alveolar bone and periodontal tissue caused by inflammation associate with periodontal tissues.
In this study, hPDLSCs were used to clarify the effects of IL-6 in periodontal tissue, especially osteogenic differentiation potential. Furthermore, a comprehensive study was conducted to elucidate the possible underlying mechanisms. Previous reports have shown that IL-6 at 10 ng/mL could benefit the proliferative potential of tendon-derived stem cells 15 , while IL-6 at 20 ng/mL enhanced osteogenic differentiation of human bone marrow   20 . Preliminarily observations of the effects of IL-6 at 10 and 20 ng/mL on the proliferation of hPDLSCs found that IL-6 at both dosages was not cytotoxic and did not influence the proliferative capacity of viability of cells. A previous study found that IL-6 had no effect on the proliferative capacity of hBMSCs or mRNA expression levels of the cell cycle regulators cyclin D1, E1, and B1 20 . The results of the present study confirmed that IL-6 does not significantly affect the proliferation and viability of hPDLSCs. However, IL-6 at 20 ng/mL significantly benefited the osteogenic differentiation potential of hPDLSCs, as demonstrated by increased ALP activity, osteogenic mRNA expression, and matrix mineralization, while the effects of IL-6 at 10 ng/mL were relatively lower. These findings are in agreement with those of previous studies showing the positive effects of IL-6 on the osteogenic differentiation potential of hBMSCs 20 , hBM-MSCs 41 , and human adipose stem cells 59 . In addition, IL-6 administration with recombinant human bone morphogenetic protein-2 and absorbable collagen www.nature.com/scientificreports/ sponge implantation enhanced ectopic bone formation in a rat model, although extensive adipogenic differentiation also occurred 20 . In a previous study, treatment of stem cells isolated from human exfoliated deciduous teeth (SHEDs) with IL-6 at 10 ng/mL enhanced stemness mRNA marker expression and osteogenic differentiation without influencing the capacity for adipogenic and neurogenic differentiation 25 . This finding suggests that IL-6 promotes osteogenic differentiation of various MSCs, depending on the cell type and study model. To achieve desirable therapeutic outcomes for oral and periodontal diseases, the treatment regimen should not only prevent disease progression, but also enhance tissue reconstruction and regeneration 37,38 . Accordingly, advanced therapeutic regimens, such as stem cell-based bone tissue engineering, are widely used for reconstruction of oral and periodontal tissues 11,60-63 . As a potential cell resource, hPDLSCs can promote osteogenesis and reconstruction of periodontal tissue or implantation 64 due to excellent osteogenic differentiation potential and bone matrix production 25,65 . According to the International Society for Cellular Therapy 66 , hPDLSCs and MSCs www.nature.com/scientificreports/ share some common characteristics, as described in our previous reports 30,32 . The hPDLSCs in the present study were characterized by spindle-shaped morphology, mRNA markers of stemness and proliferative capacity, MSCrelated surface markers, colony-forming capacity, and the potential to differentiate into osteogenic, adipogenic, and chondrogenic lineages. Hence, owing to the efficiency of the isolation protocol and robustness of the isolated cells, hPDLSCs are a good candidate for stem cell-based bone tissue engineering. Notably, IL-6 is reported to enhance the stemness of both SHEDs and BM-MSCs, but by different mechanisms 26,40,67,68 . Therefore, further studies on the effects of IL-6 on the stemness of hPDLSCs and the underlying mechanisms are warranted. The present study is the first to report and confirm that IL-6 employed the canonical Wnt/β-catenindependent pathway via either WNT2B or WNT10B to promote osteogenic differentiation of hPDLSCs in vitro. Alternatively, IL-6 may also employ the non-canonical Wnt pathway via WNT5A. Three analytic approaches were used to confirm these findings: gene silencing, recombinant Wnt, and β-catenin stabilization/translocation. However, the mechanisms underlying osteogenic differentiation of MSCs vary among different species and tissue origins 32,69-75 , which may explain the various signaling pathways employed by IL-6 for osteogenic differentiation and stemness properties [24][25][26][76][77][78] . Furthermore, a previous study reported that IL-6 employed the extracellular signal-regulated kinase (ERK)1/2-dependent pathway to maintain the stemness of hBM-MSCs in vitro 40 , while another found that IL-6 downregulated SRY-box transcription factor 2 expression, which subsequently impaired the multipotency of hBM-MSCs with commitment toward the osteogenic lineage, as demonstrated by upregulation of osteogenic mRNA markers 68 . Additional evidence suggests that osteogenic differentiation of hBM-MSCs is, at least partially, regulated by IL-6 and the membranous IL-6 receptor (IL-6R) via activation of signal transducer and activator of transcription 3 41 . MC3T3-E1 cells seeded on hydroxyapatite showed that the combination of IL-17A and IL-6 enhanced osteogenic differentiation by increasing the osteoprotegerin/RANKL ratio 79 . Figure 10. Summary of Wnt-related signaling pathways governing osteogenic differentiation of hPDLSCs treated with IL-6. The infographic illustrates potential Wnt-related signaling pathways involved in osteogenic differentiation of hPDLSCs treated with IL-6 in vitro. IL-6 employed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B to benefit the osteogenic differentiation potential of hPDLSCs in vitro. Besides, IL-6 may rely on the non-canonical Wnt pathway via WNT5A to promote osteogenic differentiation of hPDLSCs in vitro. www.nature.com/scientificreports/ Interestingly, the relationship between IL-6 and osteogenic differentiation of MSCs via Wnt signaling has not yet been elucidated. The results of the present study showed that the canonical and non-canonical Wnt pathways were employed for osteogenic differentiation of hPDLSCs treated with IL-6 via WNT2B or WNT10B and WNT5A. A previous report mentioned that the H 2 S-donor GYY4137 enhanced murine osteoblastogenesis via Wnt signaling, as demonstrated by increased expression of Wnt16, Wnt2b, Wnt6, and Wnt10b 80 . A study of chronic arthritis of the rat temporomandibular joint found that Wnt10b expression was increased in the synovial membrane with subsequent expression of Dkk-1 via a negative feedback loop 80 . An additional report of hPDLSCs isolated from inflamed tissues found that microRNA-26a-5p enhanced osteogenic differentiation by targeting Wnt5a expression via a related downstream pathway 81 . In regard to matrix production, Wnt5a was shown to induce collagen production by hPDLSCs via periostin expression through the TGF-β1 pathway 82 .
Since the Wnt signaling plays an important role in oral and maxillofacial diseases 83 , further clarification of the mechanisms underlying the activities of IL-6 in osteogenic differentiation of hPDLSCs is especially important for management of oral and periodontal diseases as well as tissue regeneration.

Materials and methods
Study approval and patient consent. The study protocol was approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (approval code: HREC-DCU 2018/054) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects.
Cell isolation, culture, and expansion. The hPDLSCs used in this study were obtained from the healthy extracted wisdom tooth of female and male subjects, aged 18-35 years, who visited the clinic for non-orthodontic reasons. The isolation protocol was modified from previous reports 25,32,84,85 . The hPDLSCs were isolated via the tissue explant technique. Briefly, human periodontal ligament tissues were collected from the mid tooth root, placed in a 35-mm culture dish, and maintained in culture medium at 37 °C under an atmosphere of 5% CO 2 . At 80% confluence, the cells were subcultured and transferred to a 60-mm culture dish. The culture medium was changed every 48 h and the cells were subcultured to 80% confluence in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% Gibco™ Antibiotic-Antimycotic (Thermo Fisher Scientific), and 1% Glutamax (Thermo Fisher Scientific). Cells at passage 2-5 were used for the experiments.
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression levels of the stemness markers REX-1, NANOG, and OCT-4, and the cell proliferation marker Ki67.
The differentiation potential of hPDLSCs toward the osteogenic, adipogenic, and chondrogenic lineages was investigated in accordance with published protocols with slight modifications 32,69,86,87 . For osteogenic differentiation, cells were seeded into the wells of 24-well culture plates at 3 × 10 4 cells/well. After 24 h, cells were maintained for 21 days in OM supplemented with 50 mg/mL of ascorbic acid (Sigma-Aldrich Corporation, St. Louis, MO, USA), 100 nM dexamethasone (Sigma-Aldrich Corporation), and 10 mM β-glycerophosphate (Sigma-Aldrich Corporation). Osteogenic differentiation potential was assessed by Von Kossa and Alizarin Red staining for mineralization of the extracellular matrix. The mRNA expression levels of the osteogenic markers RUNX2, OSX, COL1A1, ALP, OCN, and OPN were determined by RT-qPCR. Undifferentiated cells were used as a control.
For adipogenic differentiation, cells were seeded in to the wells of 24-well culture plates (5 × 10 4 cells/well) and maintained in adipogenic induction medium containing 1 µM dexamethasone (Sigma-Aldrich Corporation), 0.1 mM indomethacin (Sigma-Aldrich Corporation), 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corporation), and 0.1 mg/mL of insulin (Sigma-Aldrich Corporation) for 72 h, followed by adipogenic maintenance medium containing 0.1 mg/mL of insulin for 24 h. The induction cycle was repeated four times. Cells were then cultured in adipogenic maintenance medium until day 28. To analyze the differentiation potential, cells were stained for 1 h with Oil Red O solution (Sigma-Aldrich Corporation). Intracellular lipid droplets were observed under a microscope. The mRNA expression levels of the adipogenic mRNA markers LPL and PPRγ were measured by RT-qPCR.
For chondrogenic differentiation, cells were seeded into the wells of 24-well culture plates (3 × 10 4 cells/well) and maintained for 21 days in chondrogenic induction medium supplemented with 1% l-glutamine (Thermo Fisher Scientific, Waltham, MA, USA), 1% Gibco™ Antibiotic-Antimycotic (Thermo Fisher Scientific), 50 mg/mL of ascorbic acid (Sigma-Aldrich Corporation), 40 mg/mL of l-proline (Sigma-Aldrich Corporation), 0.1 µM dexamethasone, 1% insulin-transferrin-selenium (Thermo Fisher Scientific), 10 ng/mL of TGF-β3 (Sigma-Aldrich Proliferation and live/dead assays. Cell proliferation was evaluated using the alamarBlue™ assay. Briefly, cells were incubated in culture medium supplemented with 5% alamarBlue™ (Invitrogen Corporation) for 3 h. The spectrophotometric absorbance of each sample was recorded at wavelengths of 570 and 600 nm for reduced and oxidized spectrums, respectively. The percentage of reduction was calculated in accordance with the manufacturer's protocol. Cell viability was assessed by staining with calcein-AM (live cells-green fluorescence) and propidium iodide (dead cells-red fluorescence) and evaluated with a fluorescent microscope (ApoTome.2; Carl Zeiss Microscopy, LLC, Thornwood, NY, USA). ALP activity. ALP activity was observed at days 14 and 21 after osteogenic induction, as described previously 32,69,70 . hPDLSCs were lysed in lysis buffer containing 0.1% Triton X-100, 1 M Tris-HCl, and 5 mM MgCl 2 . Lysates were incubated with p-nitrophenol phosphate (Life Technologies, Carlsbad, CA, USA), 2-amino-2-methyl-1-propanolol (Sigma-Aldrich Corporation), and 2 mM MgCl 2 for 15 min at 37 °C. The reactions were terminated by the addition of 0.1 M NaOH. Then, absorbance was measured at a wavelength of 410 nm. The total protein concentration was measured with a Qubit™ Protein Assay kit (Thermo Fisher Scientific). ALP activity is presented as U/mg of protein.
Mineralization assay. Von Kossa and Alizarin Red staining was used to examine mineral deposition at days 14, and 21 after osteogenic induction, as described in a previous report 69,86 . For Von Kossa staining, hPDLSCs were washed with PBS, fixed with cold methanol at 4 °C for 15 min, washed with deionized (DI) water, and incubated with 5% silver nitrate solution (Sigma-Aldrich Corporation) under ultraviolet light for 30 min. Afterward, cells were washed with DI water and then mixed with 5% sodium thiosulfate (Sigma-Aldrich Corporation) for 5 min at room temperature (RT) to remove unreacted silver. Sodium thiosulfate was rinsed with DI water. Phosphate-deposited mineralization was observed under an inverted microscope as dark brown-black nodules.
For Alizarin Red S staining, hPDLSCs were washed with PBS, fixed with cold methanol for 15 min at 4 °C, washed three times with DI water (pH 4.2), and stained with 2% Alizarin Red S solution (Sigma-Aldrich Corporation) for 5 min at RT. Afterward, cells were washed 2-3 times with DI water (pH 4.2) to remove excess stain. Calcium-deposited mineralization was observed as a red color under an inverted microscope.